TOK-1, a Novel p21Cip1-binding Protein That Cooperatively Enhances p21-dependent Inhibitory Activity toward CDK2 Kinase

Abstract

A p21Cip1/Waf1/Sdi1 is known to act as a negative cellcycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a twohybrid system. Two splicing isoforms of TOK-1, TOK-1␣ and TOK-1␤, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1␣, but not TOK-1␤, directly bound to the C-terminal proximal region of p21, and both were expressed at the G 1 /S boundary of the cell cycle. TOK-1␣ also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1␣ enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1␣ is thus thought to be a new type of CDK2 modulator.Cell-cycle movement is known to be coordinately regulated by several combinations of cyclin-cyclin-dependent kinase (CDK) 1 complex and their inhibitors. INK4 family proteins, including p15, p16, p18, and p19, inhibit CDK4/CDK6, and Cip/Kip family proteins, including p21, p27, and p57, inhibit all of the CDKs (see recent reviews, Refs. 1-3 and references therein). A cDNA of p21Cip1/Waf1/Sdi1 was cloned independently by different three procedures: Cip1 (4), a cyclin-dependent kinase 2 (CDK2)-binding protein; Waf1, p53-inducible protein (5); and Sdi1, a senescent-inducible protein (6). The p21 gene is induced dependently or independently by p53 in cells after various stresses. p53-dependent p21 induction occurs by x-ray or UV irradiation, which causes DNA damage, and by heat shock or osmotic shock to cells (5). Nutrition starvation, contact inhibition, terminal differentiation, or aging of cells triggers p53-independent p21 induction that is brought about by transcription factors, including STAT family protein (7), C/EBP␣ (8), MyoD (9, 10), and vitamin D3 receptor (11). In any case, p21 induces cell cycle arrest, thus inhibiting CDK activity necessary for Rb inactivation.In addition to binding of CDK-cyclin to the N-terminal region of p21 (12-16), a variety of proteins were found to bind to the C-proximal region of p21. DNA replication activity of DNA polymerase ␦ is inhibited by p21 by binding to a proliferating cell nuclear antigen (PCNA), indicating that p21 also has the function of stopping the cell cycle during the S phase (12,13,15,(17)(18)(19)(20). Furthermore, SET/TAF1 (21), human papilloma virus E7 (22, 23), c-Myc (24), and calmodulin (25) are reported to bind to the C-terminal region of p21. The molecular mechanisms explaining the versatile functions of p21, however, remain to be determin...

Basic fibroblast growth factor (bFGF)-induced cell death is mediated through a caspase-dependent and p53-independent cell death receptor pathway

“…In the TC-32 cells p21 levels were also increased after treatment with bFGF, suggesting a potential mechanism for pRb1 activation (Ono et al, 2000). However, this is unlikely to be the sole mechanism of pRb1 activation as bFGF also activates pRb1 in the p21 negative TTC-466 cells.…”

LYRIC/AEG-1 overexpression modulates BCCIPα protein levels in prostate tumor cells

“…Using yeast two-hybrid and mammalian cells, we identified BCCIP as an interaction partner for LYRIC/AEG-1, and suggest a direct association between the two proteins. Relatively little is known about BCCIPα, but studies to date demonstrate roles in cell cycle regulation [8,9,12,20], homologous recombination repair [10,21] and cytokinesis [11].…”

“…Potentially interesting candidates were retested individually and confirmed in the yeast system. One clone, chosen for further investigation, contained cDNA encoding amino acids 3-78 of a protein originally identified by two different groups as BCCIP, a BRCA2-interacting protein [12]and TOK-1, a p21(Cip1/ Waf1) binding protein [8]. Three isoforms have been described, BCCIPα, BCCIPβ and BCCIPc, which differ in their carboxy termini.…”

“…BCCIPα was identified as a nuclear protein [8,12], while LYRIC/AEG-1 is found in both the cytoplasm and nucleus of cultured cells [17, and personal observation]. To determine whether overexpression of LYRIC/AEG-1 alters the cellular distribution of BCCIPα, we co-transfected the proteins into DU145 cells and analyzed them by indirect immunofluorescence.…”

“…Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. regulator p21, and enhances p21-mediated inhibition of Cdk2 kinase [8]. Loss of BCCIP impairs cell cycle G1/S checkpoint activation following DNA damage [9], and in conjunction with BRCA2, BCCIP plays a role in homologous recombination repair of DNA damage [10], and contributes to maintenance of chromosome stability [11].…”